Scientists “surprised” to find that CRISPR editing tool is not as precise as previously claimed

The gene-editing tool known as CRISPR-Cas12a or Cpf1 has been viewed as a better choice than other Cas editing tools because it was believed to be more precise and less prone to making off-target cuts in DNA.

But a new paper shows that Cpf1 is not as clean or specific as touted. The researchers employed in vitro assays using a vast collection of synthesized DNA molecules containing variations on the editing site sequence. They found that Cpf1 was highly prone to making off-target single-strand cuts, or "nicks", in the double-stranded DNA molecules. Off-target double-strand DNA cuts were also found, albeit at a lower frequency than the single-strand nicks.

The findings showed that the Cpf1 tool made nicks at DNA locations containing up to four DNA base unit mismatches compared to the intended target site sequence. That means it damaged a large number of markedly off-target and clearly mismatched locations.

Crucially, as the authors point out, computer programs generally used to predict the off-target DNA cut sites of CRISPR editing tools are unlikely to pick up these types of unintended Cpf1 DNA-nicking activity.

The authors conclude that “detection of potential off-target effects due to non-specific nicking would require whole genome sequencing or specialized detection methods".

The findings, which the authors describe as "surprising", show that a deeper, genome-wide scrutiny of Cpf1's in vivo (in cells) activity is necessary when it is used to generate gene-edited organisms. Researchers need to run in vivo assays with the Cpf1 editing tool and multiple different guide RNAs to check to see if what is seen in these in vitro assays translates to the real living environment of cells.

This is vital because insertions or deletions (indels) of DNA can take place at sites of nicked DNA. These can result in unintended mutations throughout the genome of the targeted cells, potentially leading to major changes in their function.

In addition, researchers need to revisit existing Cpf1-edited organisms to check for these effects.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a’s natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA–RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA (crRNA) sequence. Our analysis further revealed robust non-specific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.